During the last year, research progress has been made along three basic lines: 1. A medium has been developed for the growth of Reuber (rat) H4 hepatoma cells under serum-free conditions. The effects of medium composition, growth-substratum, insulin, and hydrocortisone were tested. The parameters measured have been growth-rate, lactate dehydrogenase activity, serum albumin secretion rate, and phenylalanine hydroxylase activity and amount. The behavior of the cells was comparable to that seen in medium-containing serum, indicating that we are able to replace the serum requirement of this cell line. 2. We have also developed serum-free cell culture conditions in which hepatocytes isolated from adult rats maintain several differentiated functions over a period of 7 to 8 days in culture. The culture medium, the cell-substratum, and the medium hormone complement have been varied. Under our conditions, the cells do not grow nor do they appear to incorporate significant tritiated thymidine. Lactate dehydrogenase activity, phenylalanine hydroxylase activity and amount, urea cycle activity, and the rate of albumin secretion have been measured. The hepatocytes under these culture conditions retain activities comparable to those found in vivo and thus appear to be a good model for liver. 3. In investigating regulation of the pure enzyme, we have discovered that phenylalanine hydroxylase must be reduced to be catalytically active. The reduction can be effected by its tetrahydropterin cofactor in a reaction that is dependent on oxygen but independent of phenylalanine. The properties of the reduction have been investigated at length; both physical and chemical changes in the enzyme are induced by reduction. (B)